Non-Aqueous Reverse Phase Purification of Carotenes on a Small Particle Preparative Packing
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1 -: 94-, Non-Aqueous Reverse Phase Purification of Carotenes on a Small Particle Preparative Packing Kevin C. O'Connor and George J. Vella Chemical Products Division,Introduction Carotenoids are a class of naturally occurring hydrocarbons consisting mostly of C40 molecules varying in the degree of conjugated double bonds. They are ubiquitous in nature and can be found in fungi, bacteria, photosynthetic and non-photosynthetic plant tissue such as algae and seaweed. Some birds, fish, amphibians and reptiles owe their vivid colors to carotenoids. Carotenes and xanthophylls are subclasses of carotenoids; the xanthophylls are oxidized forms of the carotenes and both can be dark red to yellow in color. Figure 1 shows a few of the carotene structures and their absorbance maxima. Due to the conjugated nature of these molecules considerable interest in the carotenoids has initiated research endeavors in elucidating the role they play in photosynthetic processes, vitamin A activity in vision and more recently betacarotene's role as an anticarcinogenic agent 1. Carotenes are also used as food additives, antioxidants or are available as food supplements. Hence the need for large amounts of pure carotenoids is of paramount importance. Both analytical and preparative methods for the separation of the classes of carotenoids have been reported in the literature 2-7. These methods have utilized thin layer chromatography, as well as normal phase and reverse phase column chromatography. Each method has certain advantages and disadvantages and the conditions of elution depends on the sample matrix and the particular carotenoids to be separated or isolated. Described here is a non-aqueous reverse phase method which separates configurational carotene isomers extracted from a carrot homogenate, and also permits resolution between cis and trans isomers of beta-carotene. In addition this methodology is amenable to scale-up so' that larger quantities of purified compounds can be isolated.
2 I Figure 1 Structures and absorption maxima _ nm )* of carotenes s. Beta- Carotene V Alpha-Carotene Gamma-Carotene Delta-Carotene Zeta-Carotene Lycopene * in hexane 2
3 i, Methods Sample Preparation Raw Carrots Blend w/meoh & Filter / Carrot Pulp MeOH ( Discard ) Wash with THF Until Colorless Pulp ( Discard ) THF Concentrate Evaporate to Dryness Crude Carotenes Dissolve in 25% Toluene 75% Mobile Phase HPLC Analysis
4 Optimization of Analytical Separation The separation of the carotenoids was first optimized on a high resolution analytical column (3.9 x 300mm) packed with 4gin Nova-Pak C18 material (Figure 2 A,B). Identification of the various carotenoids in the crude mixture was confirmed by comparing relative retention times to known standards and by their characteristic UV/'Vis absorbance spectra which were obtained by using an on-line Waters 994 photodiode array detector. Spectra were collected between 200nm and 600nm and were critical in the identification of several of these carotenoids. Scale Up The optimized conditions on the analytical column were transferred to a scaling column ( 8 x 100mm, cartridge column ) packed with a small particle preparative packing, 6gm Prep Nova-Pak HR C18 (Figure 2C). A loading study was carried out to determine the maximum amount of sample this column could tolerate under these conditions without compromising resolution of the components of interest. Once established, the flow rate and maximum load on the scaling column can now be used to determine the conditions to be employed for any larger volume column packed with the same particle size and of the same length. The sample load and flow rate are increased proportionally to the cross-section of the packed bed8 as shown in Equations 1 and 2.
5 Figure 2" Scale-up of Crude Carrot Extract from Analytical to Preparative Particle Sizes Column: Walers Novo-PakTM CI8, 411m,3.9mm x 300mm Mobile Phase: CH3CN/CH3OH/THF 58/35/7 Flow Rale: 1.Oral/rain Tnmperalure' 30.0 C alphacarolono AU / cis.zelo-caroleno / / hans belacaroh:ne Onto _ Irons-zelol A 0.4 ly,pone "_ao_mnaex I A H c.bo,o co,o,o.o o4 I \_ A_JU_Ld "co \ ammaxx I / I 2A 0044t _ deha<arotone\ carotene/ I I I I! I I phyli_one o, '-_ A_ /L _ 0d-I I I I I I _ ! Colu,,,n: 8mm x loomm RodiaI-Pak" Carlridge packed with Prep Nova.PakTM CI8, 61tin Mobile Phase: CH3CN/CH3OH/IIIF 58/35/7 Flow Rale: l.oml/min TemperatuJ_: 3qOC AU oo O0 I I! - 0 I Minulos 2C
6 Scaling Equations Mass (prep) / Mass (scaling) -- r2 (prep) / r2 (scaling) ( 1 ) Flow (prep) / Flow (scaling) = r2 (prep) / r2 (scaling) ( 2 ) r = column radius If both column diameter and column length are changing, the flow rate and sample load can be changed proportionally Equations 3 and 4 below: to column volume, as shown in Flow (prep) = Volume (prep) = Length (prep) r2 (prep) ( 3 ) Flow (scaling) Volume (scaling) Length (scaling) r2 (scaling) Mass (prep) = Length (prep) r2 (prep) (4) Mass (scaling) Length (scaling) o r2 (scaling) This results in identical chromatographic performance ( retention times, peak widths and resolution ) at the preparative scale as that of the scaling column. The flow rate and the maximum sample load on the scaling column were found to be 1.0ml/min ( 4.0cm/min linear velocity ) and 1.2mg ( 1501.tl) respectively. Employing equations 1-4 the flow rates and the loads were calculated for three column cartridge configurations, packed with 6p.m Prep Nova-Pak HR C18 material, which were used in a stackable cartridge holder ( Table 1 ). Chromatography is shown in Figure 3 A,B,C. Fractions were collected during the preparative separation using 25 x 300 cartridge configuration ( Fig 3A ). 6
7 Figure 3: Preparative Separation of Crude Carrot Extract using Waters PrepPak Cartridge Columns Column: Walers Nova-PakTM CI8, 61.1m25mm x I00, 200 and 300mm Carlddges Mobile Phase: CH3CN/CH3OH/IIIF 58/35/7 Sample: Crude Carrot Exlract Temperalure: 25.0 C Pump: Walers 600E Muhisolvenl Delivery System Delection: 400nm Injeclor: Walers U6K wilh a loml loop '1 A t 25mm x 30ml/min, 4.5ml loaded \ 0.4 t,n 3 3A Fm! O.4 \4-7 Ffn8 I! I I l " I h O.4- _L_ o4- '_- ^u'_-1 Od- 25mm-- x 200mm 20ml/min, _! -- 3.Oral Ioa_ I I ' I 3B 0.A _ (/,t Au- I 25ram x' l O0mm l Oral/rain, _- I ml Ioaded_ I I T... :_C () I do SO minutes
8 I Table 1. Number of Cartridges Column Volume Flow Rate Sample Load Run Time 1 x ( 8 xl00mm )* 5.0mls 1.0ml/min 1.2mgs (150_tl) 35rain 1 x (25 xloomm ) 50.Omls lo.oml/min 12.0mgs (15001.tl) 35rain 2 x ( 25 xl00mm ) 100.0mls 20.0ml/min 24.0mgs (30001Ltl) 35rain 3 x ( 25 xl00mm) 150.0mls 30.0ml/min 36.0mgs (45001Ltl) 35rain * Scaling cartridge column ( 8 x 100mm )
9 Results and Discussion Aliquots ( gi ) of the fractions collected during the preparative separation were analyzed using the high resolution 4l.t Nova-Pak column. Samples were injected in the form they were collected i.e. without evaporation or alteration ( Figures 4-_' ). Fraction 8 was identified as phytoene based on its strong absorbance a 786nm3. This spectrum is shown in Figure 4. Fractions 1 and 2 have been tentatively identified as cis-zeta-carotene and trans-zeta-carotene based on their UV/vis spectral maxima and retention relative to the other acyclic carotenes 5 ( Figures 5 and 6. ). Fraction 3 ( Figure 7 ) was identified as alpha-carotene by its retention and spectral characteristics which were identical to those of a standard. These three fractions together with phytoene represent pure components. Fractions 4,5,6 and 7 contained approximately 80%, 90%, 65% and 30% trans-beta-carotene as the spectral characteristics were very similar to those of a trans-beta-carotene standard. These fractions also exhibited absorptions between 310 and 390nm which had three maxima at 33 lnm, 347nm and 366nm in addition to absorptions attributable to cis-beta-carotene. A comparison of the three maxima matched those of phytofluene 3,5. The amount of phytofluene and cis-beta-carotene appears to vary from fractions 4 to 7 suggesting that more than one isomer of each may be present. Several isomers are known to exist for both phytofluene and cis-beta-carotene5,6. Therefore it is suggested that fraction 4 contained predominantly trans-betacarotene with a small amount of phytofluene and cis-beta-carotene. Fraction 5 appears to be most enriched with trans-beta-carotene ( >90% ) with a small amount of phytofluene. ( Figure 8 ) Fractions 6 and 7 contained decreasing amounts of trans-beta-carotene and what may be other isomers of cis-beta-carotene and phytofluene. Yet the fact that similar spectra are observed from noncontiguous fractions suggest that these other isomers are being resolved from each other however, they coelute with other carotenes found in this mixture. 9
10 Figure 4 Analysis of Fraction 8 Containing Phytoene. Column: Waters Nova.Fak TM C18, 4Ltm, 3.9ram x 300mm,._d Mobiie Phase: CH3CN/CH3OH/THF 58/35/7 FiowRate: 1.0ml/min Temperature: 30.0 C Detectzon: 286nm n = 0 5 I /V_nufe$ Fraction 8 ( rain ) AU /A ,_ 0.0,-..- ii i _0 600 am uv/vis spectrum obtained at _eak maxima during analytical separation 10
11 Figure 5" Analysis of Fraction 1 Containing cis-zeta- Carotene. AU Coiumn: Waters Nova-Fak"rMC18, 4u.m,3.9ram x 300ram Mobile Phase: CH3CN/CH3OH/THF 58/35/7 Flow Rate: 1,0ml/min - Temaerature: 30.0 C 0.12 Detection: 400nm _ I I I I Min_es Fraction 1 ( rain) AU so 30o sso _o oo uv/vis spectrumobta,ned at peak maxima during analvrxcaj separation nrn
12 Figure 6" Analysis of Fraction 2 Containing trans-zeta- Carotene. Column: Waters Nova-_akTM C18, 4L_m.3.gmm x 300mm,-, Mobile Phase: CH3CN/CH3OH/TH.: 55/35/7... Flow Rate: i.0ml/min Temaerature: 30.0 C 0.C23 - Detection: 40Onto L mw Minutes Fractian2 ( rain ) AU t so 560 s._o 460 _.so nm uv/vis spectrumo_ta,ned at peak max,maduring analytical sedarat_on
13 Figure 7: Analysis of Fraction 3 Containing Alpha- Carotene. Coiumn: Wavers Nova-PakTM C18, 4_m, 3.9ram x 300ram AU Mobile Phase: CH3CN/CH3OH/THF 58/35/7 Temperature: 30.0 C Detection: 450nm Flow Rate: 1.0ml/min k,,. I I I I I i I Minulei Fraction 3 ( rain ) AU i I I t I 1 I I 'dv/vis spectrum obtained a_ peak maxima during analytical seoorallon rlm
14 ., ( Figure 8" Analysis of Fraction 5 Containing trans-beta- Carotene. Column: Waters Nova-PakTM C18, 4urn 3.9ram x 300ram Mobile Phase: CH3CN/CH3OH/THF 58/35/7 Flow Rote:!.0ml/min Temperature: 30.0 C DetecTion: 450nm O.04O _. ; u_ 0 5I I I0 15 I 2b I 35 I Minutes as Fraction 5 ( , UV/Vis 22.Tmin ) Aut I _ S rim UV/VJs spectrum obtained at peak maxima during anatyticai separation lh
15 Conclusion The difficulty in performing a separation of this type is that all these compounds are closely related and cannot be separated using one simple chromatographic method. The separation must be developed to isolate components of interest. An analytical method for the separation and identification of carotenes in carrot homogenate has been presented. This methodology can be used to scale to larger preparative cartridges and can be used to analyze fractions collected from the preparative separation. The large scale purification of the carotenoids using a 6gm preparative reversed phase packing material permitted the isolation of five different compounds as pure components. Nine different carotenes were tentatively identified by their spectral characteristics and relative retention times. Positive identification of these would require the aid of additional structural analysis such as NMR or mass spectroscopy 5. Alternative mobile or stationary phases may afford sufficiently different selectivities, thereby resolving compounds which coelute or to separate components not addressed here or from other sources.
16 References 1. Burton, G.W. and Ingold, K.U., beta-carotene: An unusual type of lipid antioxidant. Science 224, , (1984) 2. Neils, H.J. and De Leenheer, A.P., Isocratic nonaqueous reverse phase liquid chromatography of carotenoids. Analytical Chemistry. 55, , (1983) 3. Tan, B. and Andersson, A., Analytical and preparative chromatography of tomato paste carotenoids. J. Food Science, 53(3), , (1988) 4. Bushway, R2., Separation of carotenoids in fruits and vegetables by high performance liquid chromatography. J. Liquid Chrom., 8(81, , (1985) 5. Davies, B.H., Carotenoids In " Chemistry and Biochemistry of Plant Pigments," T.W. Edwin (Ed.) Vol. 2, pg. 38 Academic Press, Orlando FL. (1976) and references therein 6. Quackenbush F.W., Reverse phase HPLC separation of cis- and transcarotenoids and its application to beta-carotene in food materials. J Liquid Chrom., 10(4), , (1987) 7. Scalia, S. and Francis, G.W., Preparative scale reversed-phase HPLC method for simultaneous separation of carotenoids and carotenoid esters. Chromatographia, 28(3,4)_, , ( 1989 ) 8. Neue, U. Private Communication (1990) 16 P.57
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